A magnetic material or magnetic dipole will move in a magnetic field to the region of highest magnetic gradient. Magnetic gradients are broadly divided into two groups. Internal magnetic gradients are formed by inducing a magnetic field on some susceptible material placed in a magnetic field, giving rise to magnetic circuits which generate a gradient. Open field gradients are formed by magnetic circuits which exist around dipole magnets such as bar or horseshoe magnets or are formed by pole piece design, orientation or configuration. In the case of a simple rectangular bar magnet, field lines which form magnetic circuits conventionally move from North to South and are easily visualized with iron filings. From this familiar experiment in elementary physics it will be recalled that there is greater intensity of field lines nearest the poles. At the poles, the edges formed with the sides and faces of the bar will display an even greater density or gradient. Thus, a steel ball placed near a bar magnet is first attracted to the nearest pole and next moves to the region of highest gradient, typically the closest edge. For magnetic circuits any design which promotes increased or decreased density of field lines will generate a gradient. In opposing magnet designs, such as N-S-N-S quadrupole arrangements, opposing north poles or opposing south poles will have field lines which will not cross each other such that in the center of such an arrangement there will be zero field (no field lines crossing the center). From the circuits that result from North poles to each adjacent South pole such arrangements generate radial magnetic gradients.
Internal high gradient magnetic devices have been employed for nearly 50 years for removing weakly magnetic materials from slurries such as in the kaolin industry or for removing nanosized magnetic materials from solution. (See Kolm, Scientific American, Nov., 1975). Typically magnetic grade stainless steel wool is packed in a column which is then placed in a uniform magnetic field which induces gradients on the steel wool. See U.S. Pat. No. 3,676,337 to Kolm. Gradients as high as 200 kGauss/cm are easily achieved. The field gradient around a wire is inversely related to the wire diameter and the smaller the wire, the faster the gradient falls off. As will be described below, collection takes place on the wire surface when it is transverse to the external magnet field lines, but not when it is tangent to them. In using such a system, material to be separated is passed through the magnetic "filter" positioned in the external magnetic field. Next the material is washed and moved to a station outside the field where magnetic materials are removed, making the collector ready for reuse. Table I below indicates the strength of the magnetic gradient as a function of distance from ferromagnetic wires of diameters of 0.1 and 0.4 mm as calculated from Maxwell's equation. These are typical wire thicknesses for the devices described above. Note that the thinner wire has a higher gradient at the wire surface, but that the gradient drops off much more quickly.
TABLE I ______________________________________ 0.1 mm diameter wire Distance from 0.4 mm diameter wire rod surface Field Distance from rod Field (mm) Strength surface (mm) Strength ______________________________________ 0 mm 170 KG/cm 0 mm 42.5 KG/cm (rod surface) (rod surface) 0.05 mm 21.1 KG/cm 0.2 mm 12.6 KG/cm 0.10 mm 6.3 KG/cm 0.4 mm 5.3 KG/cm 0.15 mm 2.7 KG/cm 0.6 mm 1.6 KG/cm 0.20 mm 1.4 KG cm 0.8 mm 0.6 KG/cm ______________________________________
Various attempts have been made to perform continuous (non-cycle) high gradient magnetic separation. Improvements include flowthrough devices with fluctuating fields to separate the magnetic material from the non-magnetic. See U.S. Pat. No. 3,902,994 to Maxwell. Removable screens of ferromagnetic material are also well known in the art. See U.S. Pat. No. 4,209,394 to Kelland. In another device patented by Kelland, U.S. Pat. No. 4,261,815, the movement of magnetic materials from the low gradient to high gradient sides (hereafter referred to as quadrants) of wires positioned transverse to an external magnetic field was utilized to perform continuous separation of magnetic materials from tailings. The device incorporates a vertical flow chamber having wires placed therein parallel to the direction of flow. It is placed in an external magnetic field such that the field is perpendicular to the flow and wires therein. As can be visualized, magnetic material in such an arrangement will move from the low gradient sides of wires to the high gradient sides. As flow proceeds, the tailings will be unaffected by the magnetic gradient. Therefore, the high gradient quadrants will contain the original concentration of tailing and two times the original concentrations of magnetic material. After sufficient travel down the wires when separation has taken place, baffles can be positioned in a quadrant fashion to prevent the magnetic and non-magnetic quadrants from mixing and to allow collection of the appropriate quadrants. In theory this will result in either magnetically enriched or depleted feed stock. By rerunning the magnetically enriched fractions, another doubling of enrichment is achieved. Repetition of this process will result in relatively pure magnetic materials.
Another method of utilization of this same magnetic phenomenon was also devised by Kelland, et al, as described in U.S. Pat. No. 4,663,029. This patent teaches the use of a non-magnetic flow chamber adjacent at one side to a ferromagnetic rod or wire, which in the presence of a magnetic field will induce a magnetic gradient. Paramagnetic material will be attracted to the wire and diamagnetic material will be repelled from the wire, allowing collection of the magnetically unique material through ports either near or far from the wire. A similar gradation of materials by magnetic susceptibility was devised by Friedlaender, F. J., et al. and is the subject of U.S. Pat. No. 4,526,681.
A method of separation useful for cells and other fragile particles was described by Graham et al. in U.S. Pat. No. 4,664,796. This apparatus contains a rectangular chamber within a cylinder. One pair of opposing sides of the chamber are made of non-magnetic material, while the other set of opposing sides are made of magnetic material. This flow chamber is packed with a magnetically responsive interstitial separation matrix such as steel wool. The material to be separated is run through this chamber which is located in a homogeneous magnetic field. In the collection mode, the chamber is aligned in the external magnetic field such that the magnetic sides of the chamber are parallel with the magnets, thus inducing a high gradient field on the interstitial matrix in the chamber. When the chamber is in this position, magnetically labeled cells are attracted to the matrix and held, while the non-magnetic components are eluted. Upon rotation of the chamber, its magnetic sides will be perpendicular to the magnets, which will "shunt" or "short-circuit" the magnetic field, lowering the gradient in the flow chamber, and allowing the particles of interest to be removed by the shear forces of the fluid flow.
There are a variety of other internal magnetic devices whose gradient properties are used to achieve different applications. Commonly owned U.S. Pat. No. 5,200,084 teaches the use of thin ferromagnetic rods used to collect magnetically labeled cells from solution. Miltenyi (WO 90/07380) teaches the use of coated steel wool, or other magnetically susceptible material to separate cells. U.S. patent application Ser. No. 07/976,476, now abandoned by Liberti and Wang teaches an internal HGMS device useful for the immobilization of cells and subsequent sequential reactions of these cells. The teaching also allows for the observation of the immobilized cells. However, the resuspension and recovery of biological substances such as cells, which are either substantially undamaged or viable, remains a stumbling block that many recent patents have attempted to solve, but with only a limited degree of success.
External gradient magnetic configurations can also be used to collect magnetically responsive particles, particularly more magnetic ones. These external devices are so-named because there is no other component of the magnetic collector except the magnets or the pole pieces. These devices rely solely on the gradients that are created via the magnetic circuits generated by the quantity and placement of magnets and in some cases by imperfections of field lines moving through space. In a standard bar magnet, gradients exist because the magnetic field lines follow non-linear paths and "fan out" or bulge as they move from North to South. These effects create gradients of about 0.1 to 1.5 kGauss/cm in high quality laboratory magnets. These relatively low gradients can be increased by manipulating the magnetic circuits so as to compress or expand field line density. For example, if the gradient at one pole of a bar magnet is of insufficient strength, moving a second bar magnet with an identical field in opposition to the first magnet would cause repulsion between the two magnets. The number of field lines would remain the same, but they would become compressed as the two magnets were forced closer together. Thus, an increased gradient would result. The addition of magnets of opposing field to this dipole configuration to form a quadrupole could further increase the size of the region of high gradients. Other configurations such as adjacent magnets of opposing fields would also create gradients higher than those seen in a bar magnet of equivalent strength. Yet another method of increasing gradients in external field devices is by adapting the pole piece design. For example, if the configuration of a standard dipole magnet were changed by making one of the magnets into a pointed magnet, all field lines would flow towards the point, dramatically increasing the gradient around that region.
None of the effects described above are new. All have been described, used, and patented for use in various industries. For example, dipoles and quadrupoles have been used in the mining industry to separate clays and ores for decades. See U.S. Pat. No. 3,326,374 to Jones and U.S. Pat. No. 3,608,718 to Aubrey. Dipoles have also reportedly been used to prevent scale and lime build up in water systems. See U.S. Pat. No. 3,228,878 to Moody and U.S. Pat. No. 4,946,590 to Herzog. Adjacent magnets of opposing polarity have been used in drum or rotor separators for the separation of ferrous and non-ferrous pieces, such as those generated in scrap yards, an improvement over the use of electromagnets. See U.S. Pat. No. 4,869,811 to Wolanski et al. and U.S. Pat. No. 4,069,145 to Sommer et al.. Other pole piece designs are well known in the literature. See Liberti & Feeley, Proc of J.Ugelstad Conference, 1991.
External magnetic devices have also been used in the fields of cell separation and immunoassay. U.S. Pat. Nos. 3,970,518 and 4,018,886 to Giaever describe the use of small magnetic particles to separate cells using an actuating coil. Dynal Corp. (Oslo, Norway) exclusively uses simple external magnetic fields to separate the many particles which it markets for various types of cell separations. Commonly owned US patent applications Ser. Nos. 08/006,071, now U.S. Pat. No. 5,466,574, and 08/228,818, now U.S. Pat. No. 5,541,072, disclose the use of external fields to separate cells, manipulating the magnetic particles and collection devices to form monolayers of cells or other biological components. However, resuspension and recovery of collected materials still requires removal of the collection vessel from the gradient field and some level of physical agitation to accomplish this.
Turning now to the magnetic particles used in such collection devices, superparamagnetic materials have in the last 20 years become the backbone of magnetic separation technology in a variety of health care and bioprocessing applications. Such materials, regardless of their size (25 nm to 100 microns,) have the property that they are only magnetic when placed in a magnetic field. Once the field is removed, they cease to be magnetic and can normally easily be dispersed into suspension. The basis for superparamagnetic behavior is that such materials contain magnetic material in size units below 20-25 nm, which is estimated to be below the size of a magnetic domain. A magnetic domain is the smallest volume for a permanent magnetic dipole to exist. Hence, these materials are formed from one or more or an assembly of units incapable of holding a permanent magnetic dipole. The magnetic material of choice is magnetite, although other transition element oxides and mixtures thereof can be used.
Magnetic particles of the type described above have been used for various applications, particularly in health care, e.g. immunoassay, cell separation and molecular biology. Particles ranging from 2 to 5 microns are available from Dynal. These particles are composed of spherical polymeric materials into which has been deposited magnetic crystals. These materials, because of their magnetite content and size, are readily separated in relatively low fields (0.5 to 2 kGauss/cm) which can easily be generated with open field gradients. Another similar class of materials are those particles of Rhone Poulanc which typically are produced in the 0.75 micron range. Because of their size, they separate more slowly than the Dynal beads in equivalent gradients. Another class of material is available from Advanced Magnetics. These particles are basically clusters of magnetite crystals, about 1 micron in size, which are coated with amino polymer silane to which bioreceptors can be coupled. These highly magnetic materials are easily separated in gradients as low as 0.5 kGauss/cm. Due to their size, both the Advanced Magnetics and Rhone Poulanc materials remain suspended for hours at a time.
There is a class of magnetic material which has been applied to bioseparations and which has characteristics that places this type of material in a special category. These are nanosized colloids (see, for example, U.S. Pat. Nos. 4,452,773 to Molday, 4,795,698 to Owen et al, 4,965,007 to Yudelson; and U.S. patent application Ser. No. 07/397,106 by Liberti, et al) . They are typically composed of single to multicrystal agglomerates of magnetite coated with polymeric material which render them aqueous compatible. Individual crystals range in size from 8 to 15 nm. The coatings of these materials have sufficient interaction with aqueous solvent to keep them permanently in the colloidal state. Typically, well coated materials below 150 nm will show no evidence of settling for as long as 6 months and even longer. These materials have substantially all the properties of ferrofluids which might be referred to as their non-aqueous compatible cousins.
Because of their size and interaction with solvent water, substantial magnetic gradients are required to separate ferrofluids. It was customary in the literature to use steel wool column arrangements of the type described above which generate 100-200 kGauss/cm gradients. However, some years ago it was discovered that such materials must form "chains" in magnetic fields like beads on a string (markedly decreasing their Stokes' drag force) because separation can be achieved in gradient fields as low as 5 or 10 kGauss/cm. These discoveries lead to the development of devices using large gauge wires which generate relatively low gradients. Large gauge wires as well as other gradient surfaces can be used to cause ferrofluids to become deposited in a substantially uniform thickness upon collection. With the proper amounts of ferrofluid in a system, the thickness of the collected material is effectively the thickness of the magnetic colloid, meaning that a monolayer can be formed. Cells magnetically labeled can be made to easily form monolayers on macro wires or uniform gradient surfaces. See commonly owned U.S. Pat. No. 5,186,827 and U.S. patent application Ser. No. 08/006,071, now U.S. Pat. No. 5,466,574.
Many techniques used in biotechnology require processes such as identification, separation, and/or manipulation of target entities, such as cells or microbes, within a fluid medium such as bodily fluids, culture fluids or samples from the environment. It is also often desirable to maintain the target entity intact and/or viable upon separation or manipulation in order to analyze, identify, or characterize the target entities.
Identification techniques typically involve labeling the target entity with a reagent which can be detected according to a characteristic property. Entities which can be viewed optically such as cells or certain microbes, may be identified and/or characterized by using fluorescently labeled probes such as monoclonal antibodies or nucleic acids. Often the target of such probes is not accessible at the surface of the target entity or an excess of probe must be removed which requires washing steps and/or exposure to a variety of reagents facilitating the penetration of the probes. As the number of operations employed in such processes increases, a greater number of target entities are lost or no longer suitable for evaluation. Accurate microbial analyses employing such methodologies are difficult to achieve because of the small numbers of target entities involved, as well as the difficulty of washing away unbound labeling agent.
For example, to measure the absolute and relative number of cells in a specific subset of leukocytes in blood, a blood sample is drawn and. incubated with a fluorescently labeled antibody specific for this subset. The sample is then diluted with a lysing buffer, optionally including a fixative solution, and the dilute sample is analyzed by flow cytometry. This procedure for analysis can be applied to many different antigens. However, the drawbacks to this procedure become apparent when large samples are required for relatively rare event analyses. In those situations, the time needed for the flow cytometer to analyze these samples becomes extremely long, making the analysis no longer feasible due to economic constraints. In addition, intra-cytoplasmic and/or intra-nuclear analysis of cell content is difficult since multiple incubations and washing steps require prohibitively long processing times.
A particular nuisance that is often experienced when collection of magnetic materials is done with continuous flow-through devices is "piling-up" of the collected material on the inlet end of the device. This occurs because collection of magnetic material effectively extends the collection surface with collected material which in itself is magnetic. Thus collection distorts the surface and the concern over lack of uniformity of collection and of trapping of tailings become significant.
Another problem with continuous separation is that once the collector surface has filled to capacity with magnetically labeled material, the separator must be physically removed from the field or somehow demagnetized so as to remove magnetic material for subsequent reuse of the device.